Although the Bicoid morphogen (Bcd) plays a pivotal role in patterning the anterior of the Drosophila (Dm) embryo, no bed homologue has been found outside Diptera. Here, we propose that the conserved hunchback (hb), orthodenticle (otd), and caudal (cad) are the key components of an ancestral anterior patterning system whose function has been taken over by bed in Dm. We will ask whether these genes can act together in Dm to control axial patterning in the absence of bcd. We will also study these genes in the wasp Nasonia vitripenis (Nv) where a mutation in hb produces a very severe anterior phenotype, consistent with our hypothesis that hb was originally necessary for extensive control of axial patterning. The morphological similarity of Nv embryogenesis to that of Dm will facilitate comparative studies. These studies in Dm and Nv will help us understand the changes underlying the acquisition of regulatory functions by the recently evolved Bcd. In parallel, we will study the molecular function of Hb, characterizing its physical interactions with itself and with Cad identified in a yeast two-hybrid screen. Aim 1 We will continue our study of Hunchback as a morphogen, including a study of its synergy with Bcd, as well as its ability to dimerize. As Dm hb might have lost functions that have been taken over by bed, we will study in Dm the patterning properties of Nv hb and of hb genes from a phylogenetic spectrum of species. To understand Nv hb regulation, its promoter will be placed in Dm to test whether bed, hb, otd or cad controls it. Using a yeast two-hybrid screen, we have observed a molecular interaction between Hb molecules. We will investigate the function of this dimerization and address whether it is required for the ability of Hb to act as a represser, while its activation requires the interaction with Bcd (or with Otd?). Aim 2 We will study the potential of Otd as a morphogen, and test whether high levels of Otd, alone or in combination with Hb, can pattern the anterior of the embryo in the absence of bcd. We will clone Nv otd, study its expression pattern and phenotype using RNAi. and study its regulation by Nv hb, or by bcd, hb and otd when placed in Din. Finally, we will assess in Dm the patterning function of Otd proteins from species where bed does not exist. Aim 3 caudal is a posterior morphogen that must be eliminated at the anterior, through transnational control by Bcd, or by a possible direct antagonism between Hb and Cad proteins. We will study the genetics, biochemistry and mechanisms of the interaction between Hb and Cad as well as the evolution of the relative contribution of the two mechanisms in invertebrates. Our comparative studies in two model insects, using the power of Dm molecular genetics combined with the unique features of Nv, will allow us to document the ancestral anterior patterning system. We hope to reconstruct in Drosophila the ancestral mechanisms, to perform a "de-evolution" from the derived fly embryo and revive the more generic type of development in insects where bed might not have appeared to coordinate anterior patterning functions.